CA2369970A1 - Hepatitis c inhibitor tri-peptides - Google Patents

Abstract

Racemates, diastereoisomers and optical isomers of a compound of formula (I):

wherein B is H, a C, ar C10 aryl, C7-16 aralkyl; Het or (lower alkyl)-Het, all of which optionally substituted with C1-6 alkyl; C1-6 alkoxy; C1-6 alkanoyl; hydroxy;
hydroxyalkyl; halo; haloalkyl; nitro; cyano; cyanoalkyl; amino optionally substituted with C1-6 alkyl; amido; or (lower alkyl)amide; or B is an acyl derivative of formula R4-C(O)-; a carboxyl of formula R4-O-C(O)-;
an amide of formula R4-N(R6)-C(O)-; a thioamide of formula R4-N(R5)-C(S)-:or a sulfonyl of formula R4-SO2; R5 is H or C1-6 alkyl; and Y is H or C1-6 alkyl;
R3 is C1-6 alkyl, C3-7 cycloalkyl, or C4-10 alkylcycloalkyl, all optionally substituted with hydroxy, C1-6 alkoxy, C1-6 thioalkyl, amido, (lower alkyl)amido, C6 or C10 aryl, or C7-10 aralkyl;
R2 is CH2-R20, NH-R20, O-R29, or S-R20, wherein R2 is a saturated or unsaturated C3-7 cycloalkyl or C4-10(alkylcycloalkyl), all of which being optionally mono-, di-or tri-substituted with R21, or R29 is a C5 or C10 aryl or C7-14 aralkyl optionally substituted, or R20 is Het or (lower alkyl)-Het, both optionally substituted, Het or (lower alkyl)-Het; carboxyl;
carboxy(lower alkyl); C6 or C10 aryl, C7-14 aralkyl or Het, said aryl, aralkyl or Het being optionally substituted; and R1 is H; C1-5 alkyl, C3-7 cycloalkyl. C2-6 alkenyl, or C2-6 alkynyl, all optionally substituted with halogen; or a pharmaceutically acceptable salt or ester thereof.

Description

HEPATITIS C INHIBITOR TRI-PEPTIDES
FIELD of Tn~ INV~NnaN
The present invention relates tv compounds, process for their synthesis, compositions and methods for the treatment of hepatitis C virus (HCV) Infection. In particular, the present invention provides novel peptide analogs, phannaceutlcal rbmpbsitibns bontaining such analbg8 and methods for using these analogs in the treatment of HCV Infection. The present invention also provides processes and intermediates for the synthesis of these peptide analogs.
BACKGROUND OF THE INVENTION
Hepatitis C virus (HCV) is the major et(plogical agent of post-tran~f~lon and community-acquired non-A non-B hepatttls worldwide. It is estimated that over million people worldwide are infected by the virus. A high percentage of carriers become chronically Infected and many progress to chronic liver disease, sa-called chronic hepatitis C. This group is in tum at high risk for serious liver disease such 16 es liver cirrhosis, hepsrtocellular carcinoma and terminal Itver dtae~aae leading to death.
rne mechanism by which HCV est8blishrds viral persistence and causes a high rate of chronic liver disease has not been thoroughly eluddated. !t la not known haw HCV interacts with and evades the host immune system. In addction, the roles of zo cellular and humoral immune roaponaea in protadion against HCV infection and disease have yet to be:established. Immunoglobulins have been reported for prophylaxia of tranafuston-aasadated viral hepatitis, however, the Center for Disease Control does not presently recommend immunoglobulins~ treatment far this purpose. The lack of an effective protective Immune response is hampering the 25 development of a vacdne or adequate post-exposure prophylaxla. measures, ac In the near-term, hopes are firmly pinned on antiviral interventions.
Various clinical studies have been conducted with the goal of identifying pharmaceutical ager~ts~ capable of effectively treating HCV infeCtic>n in patients afflicted with chronic hepatitis C. These studies have Involved the: use of Intarferon-30 alpha, alone and in combination with other antiviral agents. Such studies have shown that a substantial number of the participants do not respond to these therapies, and of those that do respond favorably, a large propartian wero found to relapse aRer termination of treatment.
Until recently, interferon (IFN) was the only available therapy of p~~oven benefit approved in the dinic for patients with chronic hepatitis C. However the sustained rosponse rate is low, and Interteron treatment also Induces sevem sidew~tfects (i.e.
retinopathy, thyrolditis, acute pancreatitls, depression) that diminish the quality of Ilfe of treated patients. Recently, interferon in combination with ribavirin has been approved far patients non-responsive to IFN alone. However, th~~ aide effects caused by IFN are not~alleviated with this combination therapy.
Therefore, a need exists for the development of effective antivlral agerrta for treatment of HCV infection that overcomes the limitations of exlsilng pharmaceutlcel therapies.
14 HCV is an enveloped positive strand RNA virus in the Flavivirldae family.
The single strand HCV RNA genome is approximately 8500 nucleotides in lejngth and has a single open reading frame (ORF) encoding a single large polyprc~teln of about amino acids. In infected oelis, this polyprotein is deaved at multiF~le sites by cellular and viral proteaaes to produce the structural and non-structural (11S) proteins, In the ~5 cax of HCV, the generation of mature nonstructural proteins (N:>2, N$~, N84A, NS4B, NS5A, and NSSB) is effected by two viral proteases. The first one, as yet poorly characterized, deaves at the N82-NS3 junction; the second one is a serine protease contained within the N-terminal region of NS3 (henceforth referred to as NS3 protease) and mediates all the subeequgnt deavages dowrn~tream of NS3, 2o both In cls, at the NS3rNS4A cleavage site, and In traps, for the remaining NS4B, NS4B-NS5A, NSSA-NS5B sites. The NS4A protein appsnrs to serve multiple functions, acting as a cafactor for the NS3 protease and possibly assisting In the membrane localtzatlon of NS3 and other viral repltcase carnponents. The complex formation of the NS3 protein with NS4A seems neaessery to the 25 processing events, enhanang the proteolytic efficiency at all of the sites.
The NS3 protein also exhit~its nuoleos~le triphosphatase and RNA helicase activities.

is a RNA-dependent RNA polymarase that Is Involved in the replication of HCV.
A general strategy for the development of antivlral agents la to irn~ctivate virally encoded enzymes that are essential for the replication of the viru,3. In this vein, 3o patent application WO 97106804 describes the (-) enantiomer of the nucleoside analogue cytosine-1,~oxathiolane (also known as 3TC) as active: against HCV.
Thla compound, although reported as sate in previous dtnical trials against HIV and HBV, has yet to be dirrically proven active against HCV and its mechanism of action against the virus has yet to ba reported.

w7 Intense efforts to discover compounds which inhibit the NS3 protease or RNA
hellcase of HCV have led to the following disclosures:
~ US patent 5,633,388 describes heterocyclio-substltuted cart~~~m(des and analogues as being active against HCV. These compounds are directed against 6 the hel(case activity of the NS3 protein of the virus but clinical tarts have not yet been reported.
~ A phenanthr~enequinonefias been reported by Chu et al., (Tr:;t. Lett., (1996).
7~2r~-7232) to have activity against the HCV NS3 protease ir.~ vitro. No further development on this compound has been reported.
~ A paper presented ~at the Ninth International Conference on Antlvlrel Research, Urabandal, Fukyah(ma, Japan (1996) (Arrtlviral Research, (1b~98), 3D, 1, A23 (abstract 19)) reports thiazalidine derivatives to be inhibitory i:o the HCV
protease.
Several studies have reported compounds inhibitory to other serlne proteases, such as human leukocyte elastase. One family of these compounds Is reported in WO
95133764 (Hoechst Marion Roussel, 1995). The peptides disclas~ad in this application ,are morpholinylcarbonyl-benzoyl-peptide analogues that are struC~rarally d(iferont from the peptides of the present Invention.
~ WO 98!17679 from Vertex Ph~trmaceut(cals Inc. discloses fnhlbltora of asrine protease, particularly, Hepatitis C virus NS3 protease. Thess~ inhibitors are peptide analogues based on the NS5IV5B natural substrate. Although several trippptides are disclosed, all of th~se peptide analogues cpnh~in C-terminal activatt3d carbonyl function as an essential feature. These ~naloguee were also reported to be active against other serine protease and are therefore not speclflc for HCV NS3 protease.
~ HofFman LaRocha has also reported hexapept(des that are protelnase inhlbRors useful as antiviral agerns for the treakment of HCV infection. 'These peptides contain an akiehyde ar a boronlc acid at the C-terminus.
Stelnkohler et al. and Ingalllnella ef al. have published on NS~4A-4B product Inhibition (Biochemistry (1998), 37, 889J-89D5 and 8906-8911). However, the peptides and peptide analogues pr~anted do not indude not' do they lead to the design of the peptides of the present invention.
One advantage of the present invention is that it provides tripepti~9es that pre inhibitory to the NS3 protease of the hepatitis C virus.

A further ~ of one asps of the present irwantion n~slds~s in the fact that these peptides speCific~lly inhibit the NS8 protease and dv not show Sign~cant inhibitory acckivity at concentrations up to 300 pNl against other se~rine prvteases such as human leukbgrte elastase (HLE), ponane pana~eatic ela.'tase (PPE), or bovine pancreatic chymotrypaln, or cystelne proteases such as human IiYer Cathepsin B (Cat 8). .
A furthst' advantage ofithe prossrtt invsrttion is that it provides small peptides of low molecular weight that r~ney be capable of penetrating cetl membr~lne9 and may be active in cell culture and !n vhro with good pharmacokinetlc proflh~_ 8uaIWARY pF i'Ha INVEWT14N
Included In the scope of the Invention Is the aompctund of formule~ (I):
or a pharmaceutically aa:eptable salt or ester thereof.
Included within the scope of this irnrsntion fs a pham~ca) o~~nposltion Comprising an anti-hepatitis G virally effeodve amount of a compound of fomtula I, or a therapautlcally acceptabler salt or ester thereof, in admixture wfth a pharmaceutically eoceptable carrier medium or auxiliary agent.
ac An important aspect of the Invention Involves a method of treating a hepatitis C viral infection in a mammal by administering to the mammal an anti-hepatitis C
virally effeGttve amount of the compound of formula I, or a therapeutically acceptable Salt or eater thereof or a composition as described above.
Another important aspect involves a method of inhibiting the ~ep~,ation of hepatitis ~5 C virus by exposing the virus to a hepatitis C viral NS3 prote2is~d inhibiting amount of the compound of fomtula l, or a therapeutically acceptable salt or ester thereof or s composldon as described above.

Still anot(1ar aBpeCt iryvolrras fi rlletf~od of tte2~ting a hop8titis C viral infection in a mammal by administering thereto an anti-hepatitis C virally effeaJve amount of a combination of tha compound of fomlula I, or a therapeutically ac:captabla salt or eater thereof. According to one embodiment, the pharmaceutical compositions of 5 this invention comprise an additional immunomodulatory agent. Examples of additional immunomoduletory agents indude but ere not limited to, c~-, ~-, and &
interierone.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
definitions As used herein, the following defln~ions apply unless otherwise noted:
Wdh reference to the instances where (R) or (S) it used to designate the conflguratlon of a aubstltuent, e.g. R' of tha compound of formul>:~ I, the designation is done In the context of the compound and not in the context pf the substituent atone.
The natural amino acids, with exc~ption of glycine, contain a chiral carbon atom.
Unless otherwise specifically indicated, the compounds containing natural amino acids with the L-coniiguratlon are pn9ferred. However, appllcante; contemplate that when specified, some amine adds of the formula I can be Of eith~.r D- or L
configuration or can be miictures of D- and L-isomers, including r~cemlc mLxtures.
2p The designation "P1, P2 and P3" as used herein refer to the position of the amino acid residues starting from the C-temnlnus end of the peptide anavlogues and extending towards the N-terminus (i.e. P1 n:fers to position 1 frorn the C-terminus, P2: saCOnd positidrt from the C-terminus, etc.) (sae Berger A. 8~ ~ichechter L, Transactions of the Royal Society London series (1970, 8257, 249-2B4].
Tha abbreviations for the c~-amino ackls used in this application sire set forth in Table A.
TABLE A
Amno Acid Symboll 1-amlnocyclopropyl-carboxylic Acca add Alanine Ala Aspartic acid Asp Cysteine Gyg CyCldtl9xylglycine (also Chg named: 2-amino-2-ohex lacetlc said Amino. Acid Symbol Glutamic acid Olu Isoleudne Ite Leucine Leu Phenylalanine Phe Proline Pro Valine Val tent Butylglycine Tbg As used herein the tens "t-aminocyclopropyl-carbo~eylic acid" (Acxs) refers to a compound of formula_ C
H'N
OH
As used herein the term "tart butylglyclne" refers to a compound of formulae H=N
'OH
The term 'residue" with reference to an amino acid or amino acrid dertvativs means a radical derived from the corresponding a-amino acki by eliminating the hydroxyl of the carboxy group and one,hydrogen of the a amino group. For instance, the terms Gln, Ala, Gly, Ile, Ar9, Asp, Phe, Ser, Leu, Cys, Asn, Sar and Tyr retxeserrt the f0 'residues" of t_-glutamlne, L-alanine, glyclne, L-lsolaucine, L-arpinlne, L-aspardc acid, L-phenylalanine, L-aerine, L-leuclne, L-cystelne, L-asparagine, sar~ccosine and t.-tyrosine, respectively.
The term "side chain" with reference to an amino acid or amino acid residue m~ans a group attached to the cx-carbon atom of the a-amino acid. For srxarnple, the R-~5 group side chain far glycine (s hydrogen, for alanlne it 3' methyl, for valins It la isopropyl. For the spec~tc R-groups or side chains of the a-amino acids reference is made to A.L. Lehninger's text on Biochemistry (see chapker a).
The term 'halo° as used herein means a halogen substituent selected from bromo, chloro, fluoro or iodo.
2o The term 'C,_s alkyl" or'(lowar)alkyl" as used herein, either alone or in combination ~ 01102/2002 818:48 X450 682 8434 ~received with another substituent, means acyclic, straight or branched chain alkyl gubstituents containing from 1 to six carbon atoms and includes, for example, methyl, ethyl, propyl, butyl, tertbutyl, hexyl, 1-methylethyl, 1-methylpropyl, methylpropyi, 1,1-dimethylethyl.
The t$rm "C~7 cyclaalkyl" as used herein, either alone or in cornt>lnatlon with another subst(tuent, means a cydoalkyl substituent containin8 fnam three to seven carbon atoms and includes cydopropyl, cyclabutyl, cydapentyl, c:ydohexyl and cydoheptyl. This term also includes "apiro"'-cyclic group such as spiro-cyclopropys or spiro~-cydobutyl:
~ ' or The term "unsaturated cycloalkyl" Includes, for example, cyclohe,xenyl:
O'' The term "C,~,o (alkylcydoalkyl) as used herein means a cydaalkyl radical containing from three to seven carbon atoms linked to an alkyl radical, the linked radicals containing up to ten carbon atoms; for example, cydopropylmethyl, cyclopentylethyl, cydohexylmethyl, cyclohexylethyl or cydohepty4ethyl.
The term "Cz.,o alkenyl" as used herein, either atone or in combination with another radical, means an alkyl radical as detlned above contalnln~ from 2 to 10 carbon stoma, and further containing at least one double bond. For example alkenyl includes allyl 2nd vinyl.
The term "Ci.a alkanoyl" as used herein, either atone or tn combination with another radlGal, meen5 9tr81ght or ranched 1-oxoalkyl radicals cantelnini~ one to~slx carbon atoms and Includes formyl, acetyl, 1-oxopropyl (propionyl), 2-methyl-1-oxopropyt, 1-oxohexyl and the like.
26 The term "C~_6 alkoxy" as used herein, either alone or in combina~6on with another radical, means the radical -O(Ci.~ alkyl) wharain alkyl is as deflneid shave containing up to six carbon atoms. Alkoxy includes methoxy, ethoxy, propo;ry, 1-methylethoxy, butoxy and 1,1-dimethylethoxy. The latter radical is known commonly as tart butoxy.
The term "C~.~ cycloalkoxy" as used herein, either alone or in combination with snottier rodlcat, mssns a Cs~ cydoslkyl group linked to sn oxy~:n atom, such ss.
for example:
O~
The term 'Ca or Cio aryl" as used here~ln, either alone or in combination with another 8 radical, means either an aromatic manocydia group containing 6 carbon atoms or an aromatic blcydlc group containing 1o carbon atoms. For example, aryl ladudes phenyl, 1-naphthyl or 2-naphthyl.
The femn 'C~_,g aralkyl" es used herein, either alone or in comt~ination with another radical, means a C.~ or C,Q aryl as defined above linked to an alk;rl group, wherein 10 alkyl is as defined above coat~ining from 1 to B carbon atoms. C~_,6 arelkyl includes for example banzyl, butylphenyl, and 1-naphthylmethyl.
The term "amino eralkyl" as used herein, either alone or in combisnation with another rad)cal, means an amino group substituted with a C,_,a aralkyl pn~up, such as, for example, the amine aralkyl:
r~
The term "(lower alkyl)amide" as u9ed herein, either alone or in combination with another radical, means an amtda mono-substttutsd with a C,.~ alkyl, such as.
~~~
The tarot "cerboxy(lower)alkyl" as used herein, ether abne or in combination with go another radicaE, means a carboxyl group (COOH) linked through a (lower)alkyl group as defined above and includes for example butyric acid.
The term "heterocyc:le" or "H~>r' as used herein, either along or In oombinadon with another radical, means a monovalent radical derived by removal of a hydrogen from a five-, six-, or seven-membered saturated or unsaturated (including aromatic) z5 heterocycle containing from one to four heteroatoms selected from nitrogen, oxygen and sulfur. Furthermore, "Hat" as used herein, means a heteroey~le as defined above fused to are or morvs other cyds, be it a hetarocycia or ayy otY~er cyde_ examples of suitable heterocydes Include: pyrrolldine, tetrahydrafuran, thlazolldtne.
pyrrole, thlophene, dlazeplne, 1 H~Imldazale, Isaxazole, thtazale, t:etrazole, 3p piperidine, 1,4-dioxane, 4-morpholine, pyrid~e, pyrimidine, thiazC~lp(4,6-b]-pyridine, quinoline, or indole, or the foltowin8 heterocyCles:
C'N~ ~ ~s . 'rNJ , 1,N-/N, ['1Nr' The term "(lower atkyl~.Het" as used herein, means a heterocydic radical as defined above linked through a chain or branched alkyl group, wherein ai~kyl is as defined 6 above cantalnlng from 1 to C carbon atoms. F~campl~s of (lower alkyl)-Het include:
o ar The term "pharmaceutically acceptable esters as used her'Ain, either alone cc' in combination with another substituant, means esters of the compound of formula I in which any of the carboxyl functions of the molecule, but preferably the cerboxy terminus, is replaced by an alkoxycarbonyl function:

,o oR
In which the R moiety of the ester Is selected from alkyl (e.g. methyl, ethyl, n-propyl, t-butyl, rr~butyl); alkoxyalkyl (e.g. methoxymethyl); alkoxyacyl (e.d.
ecetoxyrnethyl);
aralkyl (e.g_ benzyl); aryloxyalkyl (e.g_ phenoxymethyl); aryl (e.8. phenyl), optionally substituted with halogen, C,~ alkyl or C,~ alkoxy. Other suitable prodrug esters can 1s be faund in besign of prodnrgs, Bundgaard, H. Ed_ Elaevler (195) incorporated her~with by reference. Such phermaceutlcslly acceptable eat~rs ~re usually hydrolyzed irl viv0 when injected in a mammal and tr2nsformed into th9 2icid form of the compound of formula I.
With regent to the esters des~ibed above, unless otherwise spewed, any alkyl 2o moiety present advantageously contains 9 to 16 carbon atoms, particularly 1 to 6 carbon atoms. Any aryl moiety present in such esters advantageously comprises a phenyl group.
In particular the esters may be a C~_1o alkyl ester, an unsubstitut~d benzyl aster or a ber~zyl ester substituted with at least one halogen, C,.~ alkyl, C,.~ alkoxy, nitro or 25 trifluoromethyl.
The term "pharmaceutically acceptable salt' as used herein includes those derived from pharmaceutically acceptable bases. f.'-~camples of su(table bases indude cnoline, etnanolamine and ethylenediamine. No', K+, and Ca++ salts are also contemplated to be wtthln the scope of the Invention (also see Pharmaceutical salts, Binge, S.M. et al., J. Pharm. Sci., (1977), ~, 1-19, incorporated herein by reference).
5 Preferred embodiments Accordirro to an alternate embodiment, the pharmaceutical compoaittona of this InwefrtfOn may addttlanally Comprise another anti-HCV agent. Examples of anti-HCV
agents include, ac-, S- or omega-interferon, ribevirin and arnentadine.
According to another alternate embodiment, the pharmaceutical uomposldons of this 10 invention may additionally comprise other inhibitors of HCV protease.
According to yet another aPtemate embodiment, the pharmaceutGral compositions of this invention may additionally comprise an Inhibitor of other targets In the WGV Ilfe cycle, including but not limited to, helicase, polymerasA, metfilloproteas9 or irrtam2rl ribosome entry alts (IRES).
16 The pharmaceutical compositions of this invention may be ~dministered orally, parenterally or via an implanted reservoir. oral administration or administration by injection is preferred. The phanneceuticel compositions of this invention may contain any conventional non-toxic pharmaceutically-acceptable ~:artiers, adjuvar>ts or vehides_ In some cases, the pH of th~ formulation may be adjusted with 2o pharmaceutically acceptable acids, bases or buffers to enhance the stability of the formulated compound or its delivery form. The term parenteral as used herein Includes subcutaneous, intracutaneous, Intravenous, intramuscuiar, infra-ardcular, Intrasynovlal, intrasternal, intrethecal, end Intrsleslonal Injection or Infuaton techniques.
25 The pharmaceutical compositions may be In the form of a sterile Injectable preparation, for example, es a sterile injectsble aqueous or oleaginous suspension.
This suspension may be formulated according to techniques known in the art usir~
suitable dispersir~ or wetting agents (such as, far example Tween 80) and suspending agents.
3o The pharmaceutical compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, and aqueous suspanslons and solutions. In the case of tablets for or~d use, camera which are commonly used include lackose and corn stan:h_ Lubrimting agen#, such as ma~nesium stearate, are also typically added. For oral administration in a capsule form, useful dlluents Include lactose and dried com starch. When aqueous suspensions are administered orally, the active ingredient is combined with amulsifylng and suspending agents. If desired, certain sweetenityg andlor flavoring andlor coloring agents may be added.

3 Other suitable vehicles or carriers for the above noted formulations and compositions can be found in standard phartnaoeutical texts, e.s , in "Remington's Pharmaceutical Silences", The Science end Practice of Pharmacy, 98~' Ed. Mack Publishing Company, Euston, Penn., (1995).
Dosage levels of between about 0.01 and about '100 mg/kg body weight per day, 1o preferably between about 0.5 and about 75 mglkg body weight p~sr day of the protease inhibitor compounds described herein are useful in a monotherspy for the prevention and treatment df IICv mediated disease. TypiC2illy, this pharmaceutical compositions of this invention wfH be administered from about 1 tn about 5 times per day or alternatively, as a continuous infusion. Such administration can be used as a 15 chronic or acute therapy. The amount of active Ingredlant that m~sy be combined With th8 carri6r materials to prnduce a single dosage form will vary depending upon the host treated and the particular mode of administration. A typicai preparation will contain from about 5°~to,ebout 95°h active compound (wM~_ Prcaferably, such preparations contain from about 20% to about 80% active compound.
20 As the skilled artisan will appreciate, lower or higher dpses than those recited above may be required. Specfic dosage and treatment reptmens for any particular patient will depend upon a variety of fac~on3, including the activity of the ;rpeCifiv compound ~mployed, the age, body wei~ht, ~snaral health status, sex, diet; time of administration, rate of excretion, drug combination, the severity and course of the 25 IMectlon, the patient's disposition to the infection and the judgment of the treating physidan. Generally, treatment is initiated with small dosages substantially less than the optimum dose of the peptida_ Thereafter, the dp$a~g is inCreae~ed by small increments until the optimum effect undsrthe clncumstances is reached. In general, the compound is most desirably administered at a concentration level that will 30 generally afford antivirally ePfeGtive results without causing any heumful or deleterious aide eilects_ When the compositions of this invention comprise a combination ~~f a compound of fomtula I aril one or more additional therapeutic or prophylactic agent, both the cvmpvund and the additional agent should be present at dosage levels of between about 10 to 100%, and more preferably between about 10 and 8()% of the dosage normally administered in a monatherapy regimen.
When these compounds or their pharmaceutically acceptable salts are formulated together with a pharmaceutically acxeptable carrier, the resulting composltlon may be administered In vlvo to mammals, such as man, to Inhibit HCV NS3 protease or to treat or prevent HCV virus infection. Such treatment may also be achieved using the compounds of this invention in oambinatlon with agent whid~ indudo, but are not limited to: immunomodulatory agents, such a$ a-, p-, ory-int~arferons;
other antlvlrel agents such as ribavirin, amantadine; other inhibitors of HCV NS3 protease;
~o inhibitors of other targets in the HCV Ilfe cycle, which Include but not limited to, hellcsee, polymerase, metallaprobas~, or internal ribosome antr,r alts (IRES);
or combinations thereof. Ths additional agarrts may be combined ~nrith the compounds of this invention to assts a single dosage form. Altematfvely these addhlonal agents may be separately administered to a mamma( as part of a multiple dosage form.
Accordingly, another embodiment of this (mention provides methods of inhibiting HCV NS3 protease activity in mammals by administering a compound of the formula 1, wherein the substituents are as defined above.
In a prefen-ed embodiment, these methods are useful in decreasing HCV NS3 protea9e activity in a mammal. If the pharmaceuGc~al composition comprises only a compound of this invention as the active component, such methods may additionally comprise the step of administering to said mammal an agent selE:cted from an immur~omodulatary agent, an antlvlral agent, a HCV prataase Inhibitor, ar an inhibitor of other targets in the HCV life cycle such as hellcasa, g~zlymsraae, or 26 ~ metallo pratesse or IRE8. Such additional agent may be adrninis~tered to the mammal prior to, concurrently with, or following the administration of the compositions of thin invention.
In an alternate preferred embodiment, these methods are useful ~'or Inhibiting viral replication in a mammal. Such methods are useful in treating or preventing HCV
disease. If the pharmaceutical composition comprises only a compound of this Invention as the active component, such methods may additionally comprise the step of administering to said mammal an agent ae>eated from an immunomadulatary agent, an antlvlral agent, a HCV protease inhibitor, or an inhibitor' of other targets In the HCV life cyde. Such additional agent may be administered tta the mammal prior to, concurrently with, or following the administration df the Compcisitlon acDdrding to this invention.
The compounds set forth herein may also be u8ed as l8boretory re8genta. The compounds of this invention rnay alga be used to treat or prevsni: viral oontamfnatfon of materials and therefore reduce the risk of viral infection of lebc~retory or medical personnel or patierrts who come In contact with such materials (e~.g. blood, tissue, surgical Instruments and garments, laboratory instruments and garments, and blood collection apparatuses and materials).
The compounds sat forth herein may also be used as research n:agents. The 1o compounds of this invention may also b~ used as pasftive contra) to v~idate surrogate cell-based assays or In vitro or In vlvo viral replication assays.
EXAMPLES
The present invention is illustr8ted in further detail by the following non-limiting 16 examples.
Temperatures are given in degrees Celsius- $Qlution per~ntagf!s express a weight to volume relationship, and solution ratios express a volume to volume r~elatlorrship, unless stated other'Wise. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker 400 MHz spectrometer, the chemical shifts (ii) are reported in 2o parts par million. Flash chromatography was carried out an silics~ gel (SiO~
according to Still's flash chromatography technpue (W.C. Siill et al., J. Org.
Chem., (1978), 43, 2823).
Abbrwlatlons used fn the examples include Bn: beruyl; 8oc:
terf~butyloxycarbanyl (MeaCOC(C?)}; BSA: bovine serum altxrmin; CHAPS: 3-[(3-cholarnidopropyl)-~5 dimethylarnmonio]-1-propeneaulionate; DBU: 1,8-diazsbicycto[5..4.0]undeo.7-ere;
CH2CI2= DCM: methylene chloride; DEAD: diethylazodicarboxylate; DIAD:
dllsoprapylazodicarboxylate; DIEA: dilsoprapylethylamine; DIPEx~:
dllsopropylethylarnine; DMAP: dimethylarninopyridine; DCC: 1,3-dicyclohexylcarbodiirnide; DME: 1,2-dim8thy0xyethan6~; DMF:
dinnstttylfotm8mid8;
3o DMSO: dimethylsulfoxide; DTT: dlthlathroftol or thn~o-1,4-dlmarcnpta-2,3-butanedial;
DPPA diphenylphosphoryl azide; EDTA: ethylenediarninetetraacetic acid; Et:
ethyl;
EtOH: athanvl; EtOA~ ethyl acetate; Et'O: diethyl ether, HATU: [O-7-azat~nzptriszol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate]; HPLC:
high performance liquid chromatography; MS: mesa spectrometry (MA,LDi-TOF: Matrtx Assisted Laser Disorpdon Ionization-Time of Flight, FAB: Fast Atom Bombardment);
LAN: lithium aluminum hydride; Me. methyl; MeOH: methanol; MEB: (2-{N-morphollno}ethane-aulfonic acid); NaHMDS: sodium bls(trimethylallyl)amlde;
NMM:
N-methylmorpholine; NMP: N-methylpyrrolidine; Pr: propyl; Sucx; 3-carboxypropanoyl; PNA: 4-nitraphenylamino or p-nitroanilide; TBAF: t~atra-n-butylammonium fluoride: TBTU: 2-(iH-benzotrlazole-1-y1)-1,1,3,:1-tetramethyluronium tetrafluoroborate; TCEP: tris(2-carboxyethyl) phosphine hydrochloride; TFA: trinuoroacetic acid; THF: tetrahydrofuran; TI ;:
triisopropylsilane;
TLG: thin layer chrometogrBphy; TMSE: trimethylsilylethyl; TrislHGl:
trfs(hydroxymethyl)aminomethane hydrochloride.
Syn~resls of compound of formula (1) In general, the P2-P1 Intermediate (1) neoeasary for obtaining thn compound of formula (I) can be synthesized according to the methods described in UVO
00109543.
Syni~ests of P3 fragment:
horn + N OH ~ ~O~N OH

o ~ ~ o Tetrehydr~ofuran (350 mL} was added to a flask containirr9 the cy~~opentyl carbonate (9.00 g; X9.8 mmol) and flirt lducine (8.24 g; 47.6 mrriol} rersultiny. in a suspension.
Distilled water (100 mL) was added with vigorous stirring. A small amount of solid remained undisaotved. Triethylamine (16.6 mL; 119 mmol) was then added resuklng In a homogenous solution which was stir-ed at ambient ttemperaturo.
Alter Z5 2.5 h, the THF was evaporated and the aqueous residue diluted mirth water (100 mL) and the reaction rendered basic by the addition of 9 N NaOH (25 mL - final pH
a10).
The solution was washed with ethyl ocelots (? x 200 mL) and the aqueous phase then acid~ed with 1 N HGI (ca. 70 mL - final pH <2)_ The turbid s~plutiQn wag extracted with ethyl acetate (200 + 150 mL), dried over MgS04 and evaporated to a white solid (8.88 g). This material consisted of relatively pure product contaminated with hydraxysuccinamlde (~10 mol 9~ by NMR).

2.bb g of the product was chromatographed over silica gel using ethyl acetate as eluent. NOTE: The sample was loaded with dichloromethanelethyl acetate (ca.
1:1). The dichloromethane tends to result in bath the desinxi product and the UV-actlvs contaminant runntng much more quickly (and with less separation) than 6 reflected by the tic. The product was thus obtained as a white st~Ud (2.30 g) Thiourea Gyclization O
~, _ r ~ol fN.
., MW: 882.61 174.288 707.88 Combine the crude atfa-karomoketone (3.818 ; 5.71 mmol) and the: thiourea (1.099 ;
8.28mmo1} in isapropanal (14omL). place the yellow aolutlon Into a pre-heated oil bsth of 70°C. TLC (Hexane : EtOAc ; 5 : 5) after 1.5 hrs tndicate:a reacflon Is complete and the reaction Is worKed-up. Coal to l~.'f. evaporate to dryness , dilute with EtOAc wash with saturated NaHC09 (2x), water (2x) and brine (1x), dry (MgS04), filter and evaporate to obtain the product an orange-brawn foam. At this point the crude product was flash pudtled. View product by TLC I n CHCt~ :
MeCIH ; 9 1 , however, the Flash column is done in hexane : EtOAc 7 : 3 to remove less polar impurttles, then, eventually 6 : 4 to retrieve pure product as light yellow solid (3.058 ; 78°~6). M.S.(electrospray) : 706.3 (M-H)- 708.4 (M+H)+ .
Reverse Phase HPLC Homogeneity {0.08 % TFA ; CH,CN : H~0) . 98 %.
~m 01/02/2002 I(~i6=52 X450 682 8434 ~received 1~
Dsbac and Caupllnp .
~O ew !o i N 1 N~O
o H

s ~o N ~ Q

4 Ow ~ o ,~ ow b ° a MW: 707.85 2A3.31 $33.13 Dissolve the Boo-Dipeptide (3.058 ; 4.31 mmot) in 4N HCI/dioxanB (22mL) and stir at R.T. Approximately after 30~m1n. the HCI salt preclplkates and MeOH (2mL) is added 5 to render mixture in solution. After a total of 2 hours, the reaction mixture is worked-up by evaporating to dryness. The HCI salt obtained, as an off-~nrhite saEid,1a pumped under high vacuum. Dissolve the HCI salt in dichlorom~9thane (22mL) and DIEA (3.OmL ; 17.24mmcl), add the cydapentyl- Tbg-OH (1.15q ; 4.47mmol) fpllpwed by HATIJ coupling agent (1.728 ; 4.516mmol) and stir sit room temperature 'to for 5 hours. Then; dilute imlth EtOAc wash with saturated NaHC03 (2x), water (2x) and brine (1x), dry (M9SU4), filter and evaporate to obtain the crude product as a yellow solid. At this point the cruda product was flash purMed. 1~'law product by TLC
in CHCh : MeOH ; 95: 5 , however, the Flash column is done in hexane : EtOAc 7 3 to romove leas polar impurities, then, eventually 8 : 4 to rotrieve puro product a 1b white foam (3.258 ; 90%). M.8. (eleatrospray) :831.4 (M-H)- 8:53.4 (M+H)+
855.4 (M+Ne)+ . Reverse Phase HPLC Homogeneity (0.0B % TFA ; CH3CN : H20) : 98 %.
Final Hydrolysis to Provide compound A
LIOH
qor, O .~ ~
D' IJ 11 N O
M W: 833.00 818.89 Dissolve the pure methyl aster starting material (3.24mg; 3.t39mrnol) in THF
(44mL) add MeOH (20mL) followed by LiOH (1.t33mg ; 38.9mmal) dissolved in water (25mL). Tne yellow reaction mixture is stirred far 6.6 hrs a~fMr wUch it (s worked-up.
Evaporate off the organic. portion to provide an off whfta suapenaion, dilute with EtQAC end Milli-W water prepared brine to obtain a total solution. Adjust pH
to B by the addition of 1 N HCI, separate layers and extract further with EtOAc (2x) .
Combine EtCJAG extracts , wash with Mill-Q water (2x) , Milli-W water preper~ed brine (1x), dry (Mg304), flltar and evaporate to obtain the neutral product (ZV1~ as a pale yellow-white solid (3.028; 95 % yield) .
Converalon to Na Sad:
Dissolve the neutral (ZVIn produd (1.228; 1.49mrno1) in ARCH (:30mL) , add 1 equivalent O.D1 N Na4H {14.85mL) -- no product pptatlon. Shoul~:l prxipltatlon occur additional Me~H is added till a complete solution is achieved.
Concentrate 13 the dear yellow solution , dilute with Mllli-Q water, freeze and lyophililze to obtain the product (Na salt ) as a yellow-white amorphous solid {1.248; 99 % yield ) Na Salt : MUV : 84D,9t3 C4zHaaNsOoSNa M.S.(electrospray) : 817.3 (IV~H)- 819.4 (M+H)+ 841.4 (M+Na)+ . Reverse Phase HPLC HomoganaRy (0.b6 % TFA ; CHaCN ' Hi0) ' 98 %.
'H NMR (400 MH~,DMSO-d,): ca, 8:1 mixturo of ratamers ; ~' '::8.11-7.86 (m, 4H) , T.33 (bs, 1 W) , 7.18-6.97 (m. 2H), fi.36-6.08 (m, 1 H), 5.55-5.33 (m. 1 H), 4.98 (d, J = 18.0 Hz, 1 H), 4.85 (t~s, 1 H), 4.80 (d, J = 1 O:d Hz, 1 H), 4 .50-4.41 (m, 1 H), 4.22.02 (m, 2H), 3.92 (s, 3H), 2.72-2.45 {m, 1 H), 2.5D ( under IDMSO, s, 2H), 26 2.40-2.28 (m, 1 H), 1.88-1.43 (m, SH), 1.37-1.30 (m, 1 H}, 1.30-1.12 (m, 1 H), 1.03 ~
0.90 (2x s, 9H), 0.98 & 0.94 (2x s, 9H)_ Cloning, expresalon and purification oEthe recombinant HCII NS3 probase type 1 b.
Serum from an HCV-infected patient was obtaineri through an external collaboration (Bernard wllems MD, Hbpital St-~.uc, Montreal, Canada and Dr. Donald Murphy, Laboratoire de Santb Publique du Qubbec, Ste Anne de 8ellewe~, Canada). An engineered full-length cDNA template of the HCV genome was construded from DNA fragments obtained by reverse transcriptron-PCR (RT PCR) of serum RNA and using spedflc primers selected on the balls of homology between other genotype 1 b strains. From the determination of the entire genomic sequence, a genotype 1 b was assigned to the HCV Isolate accar~dlng to the classification of Simmonds et al.
~(J. Clin. Microblol., (1993), ;~1_, p.1493-15D3). The amino add sepuence of the non structural region, N82-N84B, was shown to be greater than 93°/b identical to HCV
genotype tb (BK, JK and 483 Isolates) and 88% identical to HCV genotype 1a (HCV~1 isolate). A DNA fragment encoding the poaypnotein preausor (NS3lNS4A/NS48JNS5AlNS58) was generated by PCR and ir~traducsd Into eukaryotic expression vectors. After transient transfection, the polyprotein proaessfng madlatad by the HGV NS3 protease was darnonstrdt~ld by the presence of the.mature NS3 protein usfnQ Western blot analysis. The matters NS3 protein was not observed with expression of a polyprotein precursor containing the mutation S1165A, which inactivates the NS3 protease, aonfirrning the func:~onality of the HCV
16 NS3 protease.
The DNA fragment encoding the recombinant HCV NS3 proteasta (amino add 1027 to 1208) was Boned in the pET11 d bacterial expression vector. 'The N83 protease expnsssion In E. coli BL21(DE3)pt~ysS was induced by inarbation with 1 mM IPTG
for 3 h at 22°C. A typical fermentation (18 L) yielded approximat~3ly 100 g of wet cell z0 paste. The cells were resuspanded in lysis buffer (3.0 mUp) consisting of 25 mM
sodium phosphate, pH' 7.6, 10% glyr3erol (v~), 1 mM EDTA, 0.0196 NP-40 and stored at -80°C. Cells were thawed and homogenized following the addition of 5 mM DTT. Magnesium chloride and DNase were then added to the ~homo9enate at final concentrations of 20 rnM and ZO ~ImL respectively. After a~ 25 min incubation 2'6 et ~4°C, the homogenate was soniceted and centrifuged at 150pp x g for 30 min at 4°
C. The pH of the supernatant was then adjusted to G.a using a 1 M sodium phosphate solution.
An additional gel filtration d~frometography step wss added to the 2 gt$p purfication procedure desalted In WO ~5122885 (Incorporated herein by reference). grlefly, 30 the supernatant from the bacterial extract was loaded on a SP HfTrap column (PharmaCia) previously equilibrated at a flow.rate of 2 mUmin in buffer A (50 mM
sodium phosphate, pH 6.5, 10% glycerol. 1 mM EDTA. 5 mM DTT. 0.07°~ NP-40).
The column was than washed with bufferA containing 0.16 M NaCI and trie protease eluted by applying 10 column volumes of a linear 0.15 to 0.3 M NaCI

gradient. NS3 protease-containing fractions wars pooled and dtl~,~ted to a final NaCt concentt~tion of 0.1 M. The enzyme was further purified on ~ Hflrap Heparin column (Pherrnecia) equilibrated in buffer B (25 mM Sodium phosphate, pH 7.5, 1 D°~6 glycerol, 5 mM DTT, 0.01 % NP-40). The sample was loaded at a flow rate of 3 mUmin. The Column was then washed with buffer B containing C1.15 M NeCI at a flow rate of 1.5 mUmin. Two step washes were perfom~ed in the presence of buffer t3 containing 0.3 or 1 M. NaCI. The protease was recovered in the 0.3M NaCI
wash, diluted 3-fold with buffer B, reapplied on the HiTrep Heparin column and eluted with buffer B containing 0.4~ M NaCt. Finally, the NS3 protease-containing fractions were applied on a Superdex 75 Hit.oad' 1BIB0 column (Phatmada) equilibratAC1 in buffsrr B
containing 0.3 M NaCI. The purity at the HCV N83 pro~sae obtFrine~d from the pooled fractions was judged to be greater than 95°~ by SDS-PAG3E
followed by densitomatry analysis.
The enzyme was stored at -80°C and was thawed on ice and dil~~ted just prior to use.
Rscombtnant t-!GV NS3 pcoteaseIN54A cofactor peptide radl~~metHc assay.
The enzyme was cloned, expressed and prepared according to the protocol described in Example 36. The enzyme was stored at -80°C, thawed on Ice and diluted just prior to use in the assay buffer containing the NS4A cofactor peptide.
The substrate used for the NS3 proteasal NS4A cofactor pept)de radiometric assay, DDIVPC-SMSYTW, is cleaved between the cysteine and tt~e serine residues by the enzyme. The sequence DDIYPC-SMSYTYV corresponds to the NS5AlN55B natural cleavage alts in which the cysteine residue In P2 has been substituted for a prollne.
28 The peptide substrate DDIVPC-SMSYT1N and the tracer biotin-DDIVPC-SM$j'~I-Y]TW are incubated with the recombinant NS3 protease and the NS4A peptide cofactor KKGSWIVGRIILSGRK (molar ratio enryme: cofactor 1:100) in the absence or presence of inhibitors. The separation of substrate frt~m products is performed by adding avidtn-coated ~garose beads to the essay mixture followed by filtration. The amount of SMS['~I-YjTW product found in the filtrate allows for the calcutetlon of the percentage of substrat~ convers(on end of the F>ercente~e of inhibition.
A. R~apents Tris and Tris-WC:I (UItraPure) were obtained from Gibco-BRL. Glycerol (UItraPure), 2~
MES and BSA ware purchased from Sigma. TCEP was obCalned from Plert;e, , DMSO from Atdrich and NaOH from Anachemia.
Assay buffer: 50 mM Tris HCI, pH 7.5, 30% (w/~ glycerol, 1 mglmL BSA, 1 rnM
TCEP (TCEP added just prior to use from a 1 M stock solution in water).
5 Substrate: DDIVPCSMSY?VII, 25 ~M final concentration (from a 2 mM stock solution In DMSO stored at -20°C to avoid oxidation).
Tracer reduced mono iodinated sine 6totln DDIVPC SMS[''s'1 YjTVl1 (~-1 nM
final concentration).
HCV NS3 protease type 1 b, 25 nM anal concentration (from a attack solution In 5b 10 mM sodium phosphate, pH 7.5, 10% glycerol, 300 mM NaCI, 5 mM DTT, 0.01 °~6 NP-40).
NS4A Cofactor peptide: KKGSWtVGRIILSGRK, 2.5 pM final concentwatlon (from a 2 mM stock solution in DMSO stored at -20°C).
B. Protocol 15 The assay was performed In a 96rwell polystyrene plate from Costar. Each well contained:
20 pL substrateltracer In assay buffer;
t0 p,l. t inhibitor in 20% DMSOlassay buffer, pL N83 protease 1 bINS4 cofactor peptide (molar ratio 1:100).
Blank (no Inhibitor and no enzyme) and control (no inhibitor) v~nj also pnspared on the same assay plate.
The enzymatic re~ction was initiated by the addition of the enzymd/N34A
peptide solution and the assay mixture was Incubated far 40 min at 23°C under genbe agitation. Ten (10) pL of 0.5N NacSH wero added and 10 iuL f M MES, pH 5_8 were added to quench the enrymatic reaction.
Twenty (20) pL of avidin-coated agarose beads (punch~aed from Pierce) were added in a Millipore MADP NB5 frttnation plate. The quenched a9:~ay mixture was transferred to the filtration plate, and incubated for 80 min fit Z3'(~ under gentle agitation.
3o The plates were filtered using a Millipore MuItiScreen Vacuum Manifold Filtration app8ratus, and 40 pL pf the filtrate was transferred in an opaque 96-well plate containing 60 ~L of Scintillation fluid per well.
The filtrates were counted on a Packard TopCount instrument using a'~I-liquid protocol for 1 minute.

The % inhibition was csrlculated with the following equsrtion:
100 - [(countsh~-counts~~k)I(countse,-counta~x 1 C!o) A non-linear curve flt with the Hill model was applied to the inhibition-ooncentratlon date, and the 50% effective coneentratian (ICS) was calculated try the use of SAS

6 software (Statistical Software System; SAS Institute, Inc. Cary, IVLC.).
Full-length I~S3-N94A heterodimer prot~eln essay Tha NS2-NS58-3' now coding region was cloned by RT-PCR Into the pCR~3 vector (Invftrogen) using RNA extracted from the senrm of an HCV genotype 1 b Infected individual (provided by Dr. Bernard Wrllems, Hopital St-Luc, Montreal, 4uebec, Canada). The NS3-NS4A DNA nagion was ttren subcloned by PC:R into the pFastBac~ HTa baculovirua expression vector (GibcoIBRL). Th~~ vector saquenoe indudes a region encoding a 28-residue N-terminal sequence which contains a hexahi5~tidine tag. The Bao-to-BacTM baculovirus expression system (GibGOBRL}
15' was used to produce the rocomblnant baculovlnrs. The full length mature NS3 and NS4A heterodimer protein (His-NS3-NS4AFL) was expressed by infecting 10°' Sf21 ceualmL with the rocombinant baculovirus at a multlplichy of infecxian of 0.1-0.2 at 27°G. 'fhe inixtad cukure was harvested a5 to 84 h later by oer~trlfugatlor, at 4°G.
The cell pallet was homogenized in 50mM NaPb4, pH 7.5, 40°~ ~llycarol (wlv), 2mM
p-mercaptoethanol, in prosence of a cocktail of protease Inhibitors. His-NS3-NS4AFL waS then extracted from the cell Iysate with 1.6°~b NP-4C~, 0.5%
Triton X-100, 0.5M NaCI, and a DNase treatment. After uttraceMrifugatian, the soluble extract was diluted 4fold and bound on a Ph~armacia Hi-Trap Ni-~:helatin9 column.
Tha His-NS3-NS4AFL vuas eluted in a X90°~, pure form (as Judged by SDS-pAGE), 2s using a 50 to 400 mNt Imidazole gradient. Tha His-NS3-NS4AFL was stored at -80°
C In 50 mM aadlum phosphate, pH 7.5, 10% (wIv) glycerol, 0.5 NI NaGI, D.25 M
imidezole, 4.1'16 NP-40. It wee thawed on ice end diluted just prior to use.
The protease activity of His-NS3-NS4AFL was assayed In 50 mNl Tris-HCI, pH
$.0, 0.25 M sodium strafe, ~0.01°No (wlv) n-dodecyl-[3-D-maltoslde, t mM
TCEP, Flva (5) pM of the Internally quenched substrate and~ranllyI~DDIVPAbu[Ci;O}-O]-AMY(3 NOs}TW-OH In presence of various concentrations of Inhibitor w~:re Inarbeted with 1.5 nM of His-NS3-NS4AFL for 45 min at 23°C. The final DMSO
concentration did not exceed 5.25%. The reaction was terminated with the addition of 1M MES, pH
5.8. Fluorescence of the N-terminal product was monitored on a I'erkin-Elmer LS-50B fluorometer equipped with a 96-well play reader (exdtatton wavelength: 325 nm; emission wavelength: 423 nm).
The 96 inhibition was calculated with the following equation:
10U - C(~unt9mn-countsb,"~H(countss,-counta~""k)x 100j b A non-linear curve fit with the Hill model was applied to the inhibition-concentration data, and the 506 effective concentration (ICso) was calculated key the use of SAS
soffinrare (Statistical Software System; SAS Institute, Inc. Gary, Mi.C.).
N83 Protease Cell-based assay tt) This assay was done with Huh-7 cells, a human call line derived from a hepatoma, co-tr8nsfeded with 2 DNA conetructe:
- one expressing a polyprotein comprising the HCV non~structurFil proteins fused to tTA in the following order: NS3-NS4A-N84B-N85A-tTA (called NB3);
the other expreaslnA the reporter protein, secreted alKallne pho~aphatase, under the 15 control of tTA (called SFJ~IP).
The palyprotein must be cleaved by the NS3 protease far the maturer proteins to be released. Upon release of the mature proteins, it is t~elieved that the vlrsl proteins will form a cOntplax at the memtNanB of the endapla8mic 1'etiCUlu~'r1 While tTA will migrate to the nucleus and trsnrssetlv0te the SF..AP gene. Therefore, reduction of zo NS3 prnteolytic activity should lead to nsductian of maturm tTA levels and concomitant dsaeaae In SEAP ac~tlvity.
To control for ether effects of the compounds, a parallel transfection was done where a construct expressing tTA alone (caked tTA) was cc tranf:fected with the St=AP construct such that SF~AP activity is independent of NS3 proteolytic activity.
2s Protocol at the assay: Huh~7 cells, grown in CHs-SFMtI + ~ p°,6 ~'CS
(fetal calf serum), were co-transfected with either NS3 arid SF-AP or tTA arid SEAP, u~lng the FuGene protocol (Ho~hringer Mannheim). After 6 h at 37', the cs:lls were washed, trypsinlzed and plated (at 84 000 cellslwetl) In 96-well plates comalnlng a range of concentrations of the compounds to be tested. After a 24-h incut~ation period, an 30 aliquot of the medium was drawn and the SFJ~P activity in this aliquot was measured with the Phoapha-Ltght kit (Tropix).
Analysis of th6 parGerrt inhibition of SEAP activity with respect to compound concentration was performed with ~e 5AS software to obtain the EC~o.
The toxicity of the confound (fC~) was then assessed using they M7T assay es follvvra:
20th of a MTT solution (5mglml medium) was added per wall arni incubated at 3T°
far 4 hrs;
the medium was nsmoved and 50 ~sl of 0.01N HCI + 10% Triton:C-100 was added;
S after shaking at RT for at least i hr, the OD of each well was reeHi et 585 nm wavelength.
The TC~ was calculated In the same way as the EGG.
$i~~ a~Ys ' , The specificity of the compounds was determined against a variety of S9rir~o proteases: human leukocyte alastase, porcine pancrsatlc elastriae and bovine pancreatic arrchymotrypsln and one cysteine pn~tease; human INer cathspsln B.
In all cases a 9B-well plats format protocol using a cvlarimetric p-nitroaniline (pNA) substrate specific for each enryme was used, Each assay Inducted a 1 h enryrnt-Inhibitor pre-incubation at 30°C followed by addition of substrato and hydrolysis to .d30°!o conversion as measured an a UV Thermoma~ mlcxoplatE~ reader.
Substrate conoantrat)ons were kept as low as poaslbls compared to K~,, to reduce substrate competition. Compound concentrations varlsd from 300 to 0.06 I,eM dependir~ on their potency. .
2o The final coriditians for each assay ware as follows:
50mM Trls-HCI pH 8, 0.5 M NasS04, 50 mM NaCI, 0.1 mM EDTA, 3% DMSO, 0.01 % Tween-20 with;
~ [100 pM SuCC-AAPF-pNA and 280 pM ~-chymatrypsin~, [133 L~M SucwAAA-pNA
and 8 nM porane elastaseJ, [i3S pArl Suco-AAV-pNA and 8 nM leukocyte 25 elastase]; or . [100 mM NaNPO, pH 8, 0.1 mM EDTA, 3% DM$d, 'tmM TC1_p, 0.01% Twesn-20, 30 ~M Z-FR-pNA and 5 nM cathep~sin B (the stock enzyrlle was activatdd in buffer containing 20 mM TCEP bsforo uae)j.
A representative example !s summarized below for pordne pana~ea6c elastase:
30 In a polystyrene flat-bottom 96-well plate were added using a BiOmek liquid Itfirltll8~
(t3edcman):
~ 40 ~L of assay buffer (50 mM Trls-HCI pH 8, 50 mM NaGI, 0.1 mM EDTA);
~ 20 I,~L of enzyme soluttan (50 rinM Trig-HCI pH 8, 50 mM N.CI, 0.1 mM EDTA, 0.02% Tween-20, 40 nM porcine pancreatic elastese); and ~ 20 ~L of Inhibitor solution (50 rnM Trls-HC1, pH 8, 50 mM NaC:I, 0.1 'rnM
EDTA, 0.02% Tween-20, 1.5 mM-0.3 I,~M inhibitor, 15% vlv DMS~).
After 80 min pre~incubation at 30°C, 20 p1. of substrate solution (54 mM Tris-HCI, pH 8, 0.5 M NazSO,,, 50 mM NaCI, 0.1 mM plSTA, 665 pM Suc~AAA~pNA) wero 8 added to each well and the reaction was further incubated at 30°C for 60 min after which time the ebsorbance was read on the UV Thermomax~ plate reader. Rows of wells were allocated for controls (no Inhlbltor) and for blanks (no inhibitor and no enryme).
The sequential 2 fold dilutions of the inhibitor solution were p9rform6~d on a separate 1o plate by the laquid handler using 50 mM Trls-HCI pH 8, sa mM Nt~CI, 0.1 mM
EDTA, Q.02°~6 Tween-20, 15% DMSO. All other specificity assays were performed in a similar fashion.
The peroentsge of inhibition was calculated using the formula:
[1-((UYnh-UVblank)I(Wctl-UVbiank))j x 100 15 A nQn-linear curve fit with the Hill model was applied to the inhibil:ion-concentration data, and the 5096 eifectlve concentration (IC6o) was calculated Ey the use of SAS
software (Statistical Software System; SAS Institute, Inc., Cary, P~.C.).
The compound of formula (I) ig hl~hly active against the HCV NS3 protease when 2o assessed according to~the examples above. It Is selective in that It doa not Inhlblf any of the speclflctty assays.
It also shows good phannacokinstlc properties such as detedabla plasma levels in the r'at at 1 hour after an oral dose of 5 mglkg.

Claims (11)

1. A compound of formula (I) or a pharmaceutically acceptable salt or ester thereof.

2. A pharmaceutical composition comprising an anti-hepatitis C virally effective amount of a compound of formula 1 according to claim 1, or a therapeutically acceptable salt or ester thereof, in admixture with a pharmaceutically acceptable carrier medium or auxiliary agent.

3. A method of treating a hepatitis C viral infection in a mammal by administering to the mammal an anti-hepatitis C virally effective amount of the compound of formula I according to claim 1, or a therapeutically acceptable self or ester thereof.

4. A method of treating a hepatitis C viral infection in a mammal by administering to the mammal en anti-hepatitis C virally effective amount of the composition according to Claim 2.

5. A method of inhibiting the replication of hepatitis C virus by exposing the virus to a hepatitis C viral NS3 protease inhibiting amount of the compound of formula I
according to Claim 1, or a therapeutically acceptable salt or ester thereof.

6. A method of treating a hepatitis C viral infection in a mammal by administering thereto an anti-hepatitis C virally effective amount of a combination of the compound of formula 1 according to claim 1, or s therapeutically acceptable salt or ester thereof with another anti-HCV agent.

7. A method according to claim 6, wherein said other anti-HCV agent is selected from the group consisting of: .alpha.-, .beta.- or omega-interferon, ribavirin and amantadine.

8. A method according to claim 8, wherein said other anti-HCV agent comprises an inhibitor of other targets in the HCV life cycle, selected from: helicase, polymerase, metalloprotease or IRES.

9. Use of an anti-hepatitis C virally effective amount of the compound of formula I
according to claim 1, or a therapeutically acceptable salt or ester thereof for the preparation of a composition for treating a hepatitis C viral infection in a mammal.

10. Use of a hepatitis C viral NS3 protease inhibiting amount of the compound of formula I according to claim 1, or a therapeutically acceptable salt or aster thereof for the preparation of a composition for inhibiting the replication of hepatitis C virus.

11. Use of an anti-hepatitis C virally effective amount of a combination at the compound of formula I according to claim 1, or a therapeutically acceptable salt or ester thereof, and an interferon for the preparation of a composition for treating a hepatitis C viral infection in a mammal.